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. 2009 May 29;284(22):15097-106.
doi: 10.1074/jbc.M900078200. Epub 2009 Mar 18.

The structure of an interdomain complex that regulates talin activity

Benjamin T Goult  1 , Neil Bate, Nicholas J Anthis, Kate L Wegener, Alexandre R Gingras, Bipin Patel, Igor L Barsukov, Iain D Campbell, Gordon C K Roberts, David R Critchley
Affiliations

The structure of an interdomain complex that regulates talin activity

Benjamin T Goult et al. J Biol Chem. .

Abstract

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.

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Figures

FIGURE 1.
FIGURE 1.
Structure of the talin 1655–1822 rod domain. A, schematic representation of the domain structure of talin indicating the relative position of the ligand binding sites and the boundaries of the various head and rod domains. B, sequence alignment of human talin1 residues 1655–1822 with the corresponding regions of other talins. C, superimposition of the 20 lowest energy structures consistent with the NMR data. Only the structured region of 1655–1822 is shown, not the disordered N terminus. D, ribbon drawing of a representative low energy structure showing the overall topology of the five-helix bundle. E, map of the surface charge of the domain.
FIGURE 2.
FIGURE 2.
Mapping the F3 FERM subdomain binding site in talin 1655–1822. A, 1H,15N HSQC spectra of 100 μm 15N-labeled talin 1655–1822 rod domain in the presence of the F3 FERM subdomain at ratios of 1:0 (blue), 1:0.5 (yellow), 1:1 (green), and 1:1.5 (black). B, chemical shift of the resonance of Glu-1770 of talin 1655–1822 as a function of the concentration of F3. The fitted curve of this residue (shown in red) yields a Kd of 9 (± 0.8) μm. C, weighted shift map obtained from the 1H,15N HSQC spectra of talin 1655–1822 on the addition of F3. D and E, residues in talin 1655–1822 that are significantly perturbed following the addition of F3 are highlighted on the talin 1655–1822 structure. The most significant shifts (>0.2 ppm) are shown in dark blue, and the residue numbers are labeled; smaller perturbations (>0.07 ppm) are shown in light blue. F, residues in talin F3 ((30) PDB 2H7D) that are significantly perturbed following the addition of talin 1655–1822 are highlighted on the F3 structure.
FIGURE 3.
FIGURE 3.
Structure of the complex between talin F3 and talin 1655–1822. A, ribbon representation of the model of talin F3 (red) in complex with talin 1655–1822 (pale blue) obtained using the HADDOCK approach. B, close-up of the interface showing Tyr-377 of F3 and its close proximity to Leu-1680, Val-1683, Met-1759, and Leu-1762 of talin 1655–1822. C, close-up of the interaction of the positively charged activation loop of F3 with the negatively charged residues on talin 1655–1822.
FIGURE 4.
FIGURE 4.
Identification of mutants that disrupt the head-tail interaction. A–C, weighted shift map obtained from the 1H,15N HSQC spectra of talin 1655–1822, or mutants thereof, on the addition of talin F3. A, wild-type (WT) talin 1655–1822. B, E1770A talin 1655–1822. C, T1767E talin 1655–1822. D, dissociation constants for the binding of wild-type talin 1655–1822 and various mutants thereof to F3. E, dissociation constants for the binding of wild-type talin 1655–1822 to various F3 FERM subdomain mutants (obtained from analysis of the chemical shift changes of residues 319, 324, 326, 352, 359, 366, 373, and 378 in the 1H,15N HSQC spectra of F3, or mutants thereof, on the addition of talin 1655–1822).
FIGURE 5.
FIGURE 5.
The talin 1655–1822 rod domain competes with the β3-integrin tail for binding to the talin F3 FERM subdomain. A, structure of the talin F3-integrin tail complex ((30) PDB 2H7D). Talin F3 is shown in red; the integrin tail is shown in yellow, and the membrane is shown by a black line. B, the F3-talin 1655–1822 complex oriented as in A, the binding site for the integrin tail, and the membrane association site within the activation loop of F3 are masked by the talin rod domain. Talin F3 is shown in red, and talin 1655–1822 is shown in cyan. C, weighted chemical shift maps of 15N-labeled β3-integrin tail upon the addition of talin 1655–1822 (red), talin F3 domain (blue), or both (green). The upper graph shows results for the wild type rod domain, and the lower graph shows results for the E1770A talin rod domain mutant. Gray bars correspond to peaks that broaden severely and could not be tracked.
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